A mechanism of lysosomal calcium entry.

Lysosomal calcium (Ca2+) release is critical to cell signaling and is mediated by well-known lysosomal Ca2+ channels. Yet, how lysosomes refill their Ca2+ remains hitherto undescribed. Here, from an RNA interference screen in Caenorhabditis elegans, we identify an evolutionarily conserved gene, lci-1, that facilitates lysosomal Ca2+ entry in C. elegans and mammalian cells. We found that its human homolog TMEM165, previously designated as a Ca2+/H+ exchanger, imports Ca2+ pH dependently into lysosomes. Using two-ion mapping and electrophysiology, we show that TMEM165, hereafter referred to as human LCI, acts as a proton-activated, lysosomal Ca2+ importer. Defects in lysosomal Ca2+ channels cause several neurodegenerative diseases, and knowledge of lysosomal Ca2+ importers may provide previously unidentified avenues to explore the physiology of Ca2+ channels.

The evolution of organellar calcium mapping technologies.

Intracellular Ca2+ fluxes are dynamically controlled by the co-involvement of multiple organellar pools of stored Ca2+. Endolysosomes are emerging as physiologically critical, yet underexplored, sources and sinks of intracellular Ca2+. Delineating the role of organelles in Ca2+ signaling has relied on chemical fluorescent probes and electrophysiological strategies. However, the acidic endolysosomal environment presents unique issues, which preclude the use of traditional chemical reporter strategies to map lumenal Ca2+. Here, we broadly address the current state of knowledge about organellar Ca2+ pools. We then outline the application of traditional probes, and their sensing paradigms. We then discuss how a new generation of probes overcomes the limitations of traditional Ca2+probes, emphasizing their ability to offer critical insights into endolysosomal Ca2+, and its feedback with other organellar pools.

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis.

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

Quantifying phagosomal HOCl at single immune-cell resolution.

Neutralization of pathogens by phagocytic immune cells requires the biogenesis of a compartmentalized hotspot of reactive species called the phagosome. One of these reactive species is hypochlorous acid (HOCl), produced by the enzyme myeloperoxidase (MPO) after the phagosome fuses with the lysosome. Mapping HOCl during phagosome maturation can report on pathogen killing and offer insights into regulation of MPO activity, mechanisms of resistance and host-pathogen interactions. However, this has been difficult because of a lack of a suitable method to chemically map a transient organelle with pH fluctuations like the phagosome. Here, we detail a protocol for quantifying HOCl dynamics in phagosomes using a fluorescent DNA-based reporter. Compared to traditional methods of visualizing HOCl or measuring MPO activity, this method offers sub-cellular spatial resolution and the capacity to assay HOCl production with single cell resolution.

What biologists want from their chloride reporters - a conversation between chemists and biologists.

Impaired chloride transport affects diverse processes ranging from neuron excitability to water secretion, which underlie epilepsy and cystic fibrosis, respectively. The ability to image chloride fluxes with fluorescent probes has been essential for the investigation of the roles of chloride channels and transporters in health and disease. Therefore, developing effective fluorescent chloride reporters is critical to characterizing chloride transporters and discovering new ones. However, each chloride channel or transporter has a unique functional context that demands a suite of chloride probes with appropriate sensing characteristics. This Review seeks to juxtapose the biology of chloride transport with the chemistries underlying chloride sensors by exploring the various biological roles of chloride and highlighting the insights delivered by studies using chloride reporters. We then delineate the evolution of small-molecule sensors and genetically encoded chloride reporters. Finally, we analyze discussions with chloride biologists to identify the advantages and limitations of sensors in each biological context, as well as to recognize the key design challenges that must be overcome for developing the next generation of chloride sensors.

A DNA Aptamer for Cyclic Adenosine Monophosphate that Shows Adaptive Recognition.

As a ubiquitous second messenger, cyclic adenosine monophosphate (cAMP) mediates diverse biological processes such as cell growth, inflammation, and metabolism. The ability to probe these pathways would be significantly enhanced if we had a DNA-based sensor for cAMP. Herein, we describe a new, 31-base long single-stranded DNA aptamer for cAMP, denoted caDNApt-1, that was isolated by in vitro selection using systemic evolution of ligands after exponential enrichment (SELEX). caDNApt-1 has an approximately threefold higher affinity for cAMP than ATP, ADP, and AMP. Using non-denaturing gel electrophoresis and fluorescence spectroscopy, we characterized the structural changes caDNApt-1 undergoes upon binding to cAMP and revealed its potential as a cAMP sensor.

High-Throughput Assessment of Structural Continuity in Biologics.

We demonstrate a high-throughput chemoprinting platform that confirms the consistency in the higher-order structure of protein biologics and is sensitive enough to detect single-point mutations. This method addresses the quality and consistency of the tertiary and quaternary structure of biologic drug products, which is arguably the most important, yet rarely examined, parameter. The method described uses specific small-molecule ligands as molecular probes to assess protein structure. Each library of probe molecules provides a "fingerprint" when taken holistically. After proof-of-concept experiments involving enzymes and antibodies, we were able to detect minor conformational perturbations between four 48 kDa protein mutants that only differ by one amino acid residue.

An application of borane as a protecting group for pyridine.

Efforts to couple 4 with 12 employing base mediation are problematic due to the formation of 6. To circumvent this issue, 12 was converted to the pyridine borane complex (13). Alkylation of 4 with 13 provided 3 after removal of the borane under acidic conditions and saponification of the ester.

Efficient transamidation of primary carboxamides by in situ activation with N,N-dialkylformamide dimethyl acetals.

Two protocols for the transamidation of primary amides with primary and secondary amines, forming secondary and tertiary amides, respectively, are described. Both processes employ N,N-dialkylformamide dimethyl acetals for primary amide activation, producing N'-acyl-N,N-dialkylformamidines as intermediates, as widely documented in the literature. Although the latter intermediates react irreversibly with amines by amidinyl transfer, we show that in the presence of certain Lewis acid additives efficient acyl transfer occurs, providing new and useful methods for amide exchange. In one protocol for transamidation, the N'-acyl-N,N-dialkylformamidine intermediates are purified by flash-column chromatography and the purified intermediates are then treated with an amine (typically, 2.5 equiv) in the presence of scandium triflate (10 mol %) in ether to form in high yields the products of transamidation. In a second procedure, N'-acyl-N,N-dialkylformamidines are generated in situ and, without isolation, are subjected to transamidation in the presence of zirconium chloride (0.5 equiv) and an amine (typically 2 equiv). A variety of different primary amides and amines are found to undergo efficient transamidation using the methods described.

A synthesis of the diazonamide heteroaromatic biaryl macrocycle/hemiaminal core.

[reaction: see text] Stille coupling of an arylstannane aminal 19 with the palladium complex 23 leads to atropisomeric esters 28 and 29. Conversion to macrocycle 30 is demonstrated, a potential precursor of natural and unnatural diazonamides.